Citrus greening blight, also known as Huanglongbing (HLB), is a lethal disease in citrus plants across the world, causing significant fruit loss and death of infected trees. HLB infection affects the leaves, twigs and fruit of the tree, eventually causing the whole tree to decline. Symptoms of HLB include blotchy, mottled and yellow-veined leaves, lopsided and small, green, salty and bitter flavored fruit, twig dieback, and overall tree stunting and decline. Diagnosis of HLB is complicated by the fact that some of the symptoms of HLB are similar to symptoms of other tree ailments, including nutritional deficiency. HLB control often requires destroying infected trees, which may not even show symptoms of HLB for several years after becoming infected.
Polymerase chain reaction diagnostic testing has identified the major pathogen associated with symptoms of HLB as the bacterium Candidatus Liberibacter spp, including the Ca. L. asiaticus (LAS), Ca. L. africanus and Ca. L. americanus species. The bacteria is believed to impair nutrient transfer in a citrus tree's phloem and leaf stems, wherein the plant pathology is suspected to be caused by nutrient deficiency. Antibiotic control of the putative causative bacterium is a passive solution but an expensive means of grove management that couldnever be a plausible solution. Low replication levels of Ca. L. in citrus require high sensitivity methods to detect their presence. PCR based methods that have been employed for the detection of Ca. L. asiaticus bacterium include qualitative PCR assays (Saponari et al., 2013, J. Virol. Methods. 193(2): 478-86) and tissue-blot diagnosis (Nageswara-Rao M. et al., 2013, Mol. Cell Probes. 27(5-6): 176-83), each of which are herein incorporated by reference.
Bacteriophages or phages are viruses which infect bacteria. The majority are DNA viruses. Each phage attacks only particular species or in many cases only certain strain within the species. Structurally, a bacteriophage or phage is composed of a polygonal head consisting of DNA surrounded by a thin protein membrane and a short straight tail consisting of protein. However, spherical and filamentous phages have also been reported. The life cycle of bacteriophages includes two cycles: vegetative cycle and lysogenic cycle. In the vegetative cycle, the phage is called virulent phage, and the cycle contains the steps of attachment (adsorption), penetration, eclipse phase, replication, assembly, and release. In the lysogenic cycle, the phage is called temperate phage, and the cycle contains the steps of attachment (adsorption), penetration, eclipse phase, and then unlike the virulent phage in the vegetative cycle, the temperate phage after the eclipse phase does not replicate, instead, it is integrated into the bacterial chromosome and remains latent for a period of time. The integrated phage is now called prophage, and the bacteria carrying prophage is called lysogen. The lysogen is not lysed but grows and multiplies with prophage as a part of the chromosome. The lysogen can acquire new properties, and the prophage may revert to virulent phage and infected bacteria may be lysed. The practical applications of bacteriophages include, but are not limited to, bacteriophage typing, classification of bacteria into phage groups, phage lysogenic conversion, phage therapy, and as cloning vectors.
Bacteriophages such as those associated with Staphylococci and the intestinal commensal bacterium E. faecalis are deemed important factors in the life cycle of host bacteria that may affect the pathogenicity of the host (Deghorain and Melderen, 2012, Viruses 4:3316-35; Duerkop et al., PNAS 109(43):17621-626). Analysis of the Ca. L. asiaticus genome derived from HLB symptomatic citrus has revealed the presence of two circular phage genomes, SC1 and SC2 (Zhang et al., 2011, Molecular Plant-Microbe Interactions 24(4):458-68). Three DNA fragments (In-2.6, In-1.0 and In-0.6) of the non-cultured, bacterial-like organism (BLO) associated with citrus greening disease were cloned, and nucleotide sequence determination showed that the genome of the non-cultured BLO associated with citrus greening disease contains the nusG-rp1KAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase (Villechanoux et al., 1993, Curr Microbiol. 26(3):161-6), the entire contents of this reference is incorporated by reference herein. Complete genome of Liberibacter crescens BT-1 was sequenced and contains more genes in thiamine and essential amino acid biosynthesis, as well as contains two prophage regions (Leonard et al., 2012, Standards in Genomic Sciences 7:271-83), the entire contents of which is incorporated by reference herein.
In light of the foregoing, there still remains a need in the art for HLB diagnosis methods and eradication methods and compositions for the disease, as well as more generally diagnostic and treatment methods and compositions for pathogen-pathogenic-host mediated disease systems.